On the influence of the culture conditions in

bacterial antifouling bioassays and biofilm

properties: Shewanella algae, a case study
Martín-Rodríguez et al.
Martín-Rodríguez et al. BMC Microbiology 2014, 14:102
http://www.biomedcentral.com/1471-2180/14/102
Martín-Rodríguez et al. BMC Microbiology 2014, 14:102
http://www.biomedcentral.com/1471-2180/14/102

RESEARCH ARTICLE Open Access

On the influence of the culture conditions in

bacterial antifouling bioassays and biofilm
properties: Shewanella algae, a case study
Alberto J Martín-Rodríguez1,5*, Alejandro González-Orive2, Alberto Hernández-Creus2,6, Araceli Morales3,
Roberto Dorta-Guerra4, Manuel Norte1, Víctor S Martín1 and José J Fernández1*

Abstract
Background: A variety of conditions (culture media, inocula, incubation temperatures) are employed in antifouling
tests with marine bacteria. Shewanella algae was selected as model organism to evaluate the effect of these parameters
on: bacterial growth, biofilm formation, the activity of model antifoulants, and the development and nanomechanical
properties of the biofilms.
The main objectives were:
1) To highlight and quantify the effect of these conditions on relevant parameters for antifouling studies: biofilm
morphology, thickness, roughness, surface coverage, elasticity and adhesion forces.
2) To establish and characterise in detail a biofilm model with a relevant marine strain.
Results: Both the medium and the temperature significantly influenced the total cell densities and biofilm
biomasses in 24-hour cultures. Likewise, the IC50 of three antifouling standards (TBTO, tralopyril and zinc
pyrithione) was significantly affected by the medium and the initial cell density. Four media (Marine Broth, MB; 2% NaCl
Mueller-Hinton Broth, MH2; Luria Marine Broth, LMB; and Supplemented Artificial Seawater, SASW) were selected to
explore their effect on the morphological and nanomechanical properties of 24-h biofilms. Two biofilm growth patterns
were observed: a clear trend to vertical development, with varying thickness and surface coverage in MB, LMB and
SASW, and a horizontal, relatively thin film in MH2. The Atomic Force Microscopy analysis showed the lowest
Young modulii for MB (0.16 ± 0.10 MPa), followed by SASW (0.19 ± 0.09 MPa), LMB (0.22 ± 0.13 MPa) and MH2
(0.34 ± 0.16 MPa). Adhesion forces followed an inverted trend, being higher in MB (1.33 ± 0.38 nN) and lower in
MH2 (0.73 ± 0.29 nN).
Conclusions: All the parameters significantly affected the ability of S. algae to grow and form biofilms, as well as
the activity of antifouling molecules. A detailed study has been carried out in order to establish a biofilm model
for further assays. The morphology and nanomechanics of S. algae biofilms were markedly influenced by the nutritional
environments in which they were developed. As strategies for biofilm formation inhibition and biofilm detachment are
of particular interest in antifouling research, the present findings also highlight the need for a careful selection of the
assay conditions.
Keywords: Biofilm, Biofouling, Antifouling, Shewanella algae, CLSM analysis, Atomic Force Microscopy, Young modulus,
Adhesion forces

* Correspondence: ajmartinr@ull.es; jjfercas@ull.es

1
Institute for Bio-Organic Chemistry “Antonio González”, Center for Biomedical
Research of the Canary Islands (CIBICAN), University of La Laguna, Avenida
Astrofísico Francisco Sánchez 2, La Laguna, Tenerife 38206, Spain
5
Oceanic Platform of the Canary Islands, Carretera de Taliarte s/n, Telde, Gran
Canaria 35214, Spain
Full list of author information is available at the end of the article

© 2014 Martín-Rodríguez et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the
Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public
Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this
article, unless otherwise stated.
Martín-Rodríguez et al. BMC Microbiology 2014, 14:102
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Background
Biofouling is a colonisation process that begins from the
very same moment a material surface is immersed in sea-
water and leads to the development of complex biological
communities. This undesirable accumulation of biological
material causes severe economic losses to human activities
in the sea, from deterioration of materials, structures,
and devices, to increases in fuel consumption and loss
of maneuverability in ships [1,2]. In a simplified model,
there are four main stages in the biofouling process: i)
adsorption of organic matter onto the material surface,
creating a conditioning film; ii) arrival of the so-called
primary colonizers (bacteria and diatoms, mainly) that
form complex, multispecies biofilms; iii) settlement of
spores of macroscopic algae and other secondary colo-
nisers; and iv) settlement of invertebrate larvae [3]. Even
though it is not necessarily a sequential process, it is
generally accepted that the formation of an organic layer
and a biofilm is the first step to biofouling [4].
Since the ban on the use of organotin compounds,
particularly bis-(tris-n-butyltin) oxide (TBTO), established
by the International Maritime Organization (IMO) that
finally entered into force in September 2008, there is a
clear need for alternative antifouling compounds. We have
recently started a screening program for the search of
novel antifouling molecules. In doing so, one of the most
striking issues is the great diversity of conditions currently
employed in lab-scale assays (i.e., culture media, inocula,
incubation times and temperatures), not only when deal-
ing with biofilms, in whose case the optimal conditions
should be individually defined for each strain, but even
with planktonic cultures [5-11]. It seems evident that this
heterogeneity may lead to important differences in the
results obtained from in vitro tests. In addition, there is a
lack of studies focusing on the effect that these diverse
conditions have on the properties of marine biofilms.
Even though single-strain laboratory tests do not mimic
the real environmental conditions, in vitro models are a
useful tool for screening and comparing new products,
treatments and materials. To this end, S. algae was chosen
as model organism. Shewanella spp. are gram-negative,
facultative anaerobe rod-shaped uniflagellar bacteria world-
wide distributed in marine and even freshwater habitats
(Figure 1) [12,13]. They play an important role in the bio-
geochemical cycles of C, N and S [13] due to their unpar-
alleled ability to use around twenty different compounds
as final electron acceptors in respiration, which, in turn,
provides bacteria the ability to survive in a wide array of
environments [14]. For this versatility, shewanellae have
been focus of much attention in the bioremediation of
halogenated organic compounds, nitramines, heavy metals
and nuclear wastes [14]. Their biofilms are also receiving
increasing interest in microbial fuel cell applications due
to their excellent extracellular electron transfer capability,
Page 2 of 14
that includes direct (e.g. nanowires) and indirect mecha-
nisms (electron shuttles such as flavins) [15]. Shewanella
spp. biofilms have been found to modulate the settlement
(with inductive or inhibitory effects) of a variety of macro-
scopic algae and invertebrates such as Ulva spores [16-18],
cypris [19], mussel larvae [20], or sea urchin larvae [21].
Shewanella spp. produce omega-3 fatty acids and other
hydrocarbons, probably to increase the fluidity of the cell
membrane in cold waters –most Shewanella strains are
psychrotolerant- or as a result of a mutualist relationship
between fish and bacteria living in their intestines [14,22].
Indeed, they are being increasingly used as probiotics in
aquaculture [23,24] and, more recently, as a source of
hydrocarbon fuels [22]. Among all the members of the
shewanellae family, only S. putrefaciens and S. algae are
widely recognized to be pathogenic to human and animals,
being involved in soft-tissue infections, ear infections,
necrotising fasciitis, abscesses, bacteremia, and many other
affections [12,25-29]. However, there is increasing evidence
that point that other Shewanella species are also causative
agents of human infections [30,31]. For all these reasons,
S. algae biofilms are of great interest in bacterial fouling
studies as well as in many other fields.
In anti-biofilm assays, the nutritional requirements
that promote bacterial biofilm formation may not be the
same as those employed in antimicrobial susceptibility
testing, thus leading to the use of a different culture
medium and frequently higher inocula [32]. In order to
explore the effect of the culture conditions on the growth
and biofilm formation of S. algae, nine media and two incu-
bation temperatures were initially screened. Subsequently,
the antibacterial activity of known antifouling biocides was
determined using different media and inocula. Finally, in
order to assess exhaustively the morphological and physical
properties of S. algae biofilms developed in different media,
a detailed examination was conducted by Confocal Laser
Scanning Microscopy (CLSM) and Atomic Force Micros-
copy (AFM). Over the last few years, AFM has turned into
a powerful technique not only for studying the morphology
of soft materials such as polymers and biomaterials but
also for obtaining information about different properties
(mechanical, electrical, magnetic, etc.) of the samples.
In particular, recent advances in force distance-based
AFM (FD-AFM) have allowed researchers to register
three-dimensional quantitative images related to interesting
physicochemical properties of living cells [33,34]. PeakForce
Tapping (PFT) in liquid media is a novel, cutting edge
breakthrough in AFM that allows the imaging and quanti-
fication of the physicochemical properties associated to
every point in a 3D surface immersed in a liquid environ-
ment. This is of special interest for biological samples and
particularly for marine biofilms, so we have been able
to measure these properties directly in natural seawater.
In this article FD-AFM methods have been used to
Martín-Rodríguez et al. BMC Microbiology 2014, 14:102
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Figure 1 Tapping mode images in air of Shewanella algae adsorbed on treated polystyrene. (A) 10 × 10 μm2 bidimensional image
showing bacterial dimensions and their characteristic flagella; (B) 3.2 × 3.2 μm2 three-dimensional image with bacterial surface roughness and
flagella in detail. White arrows indicate the position of flagella.
characterise the morphology of biofilms of S. algae grown
in different nutritive media and to obtain quantitative
mapping of elastic modulus and adhesion forces of the
resulting biofilms.
Results and discussion
Influence of the culture conditions on bacterial growth
and slime production
Bacterial growth was initially checked in agar plates of
the nine culture media at 20°C, 26°C and 32°C after 24 h
in order to qualitatively assess the best range of temper-
atures. From these initial observations, the lower incuba-
tion temperature was ruled out due to poor growth.
Media with different characteristics were chosen
(Additional file 1: Table S1): Marine broth (MB) is a
widespread culture medium for marine bacteria that
contains high levels of salts as well as trace elements.
Its main difference with the Supplemented Artificial
Seawater medium (SASW) and Luria Marine Broth (LMB)
is the amount of primary sources of carbon and nitrogen,
and the trace element content [35]. Väätänen Nine-Salt
Solution (VNSS) is a complex salt-rich medium that is
frequently used in marine microbiology [36,37]. Mueller-
Hinton is the standard culture medium in antimicrobial
susceptibility tests, and often it needs to be supplemented
with salts (2%, MH2) and/or calcium and magnesium
(cation-adjusted MH2, CAMH2) to support the growth
of certain bacteria like pathogenic vibrios [38,39] and
halophilic marine strains [40,41]. Brain-Heart Infusion
and Tryptic Soy Broth were also supplemented with
2% NaCl and designed as BHI2 and TSB2, respectively.
These NaCl-supplemented rich media have been previ-
ously employed in the culture of Pseudoalteromonas and
Vibrio species [15,16]. A minimal medium (MMM) was
included to evaluate the effect of a limiting environment
Page 3 of 14
on biofilm formation. The actual starting cell density was
7.0 ± 0.8 × 105 cfu/ml.
Figure 2 shows the total cell density (A) and biofilm
biomass (B) in different media at the two selected temper-
atures. In order to determine the effect of the medium,
the temperature and the interaction on the total cell dens-
ity and biofilm formation, ANOVA tests were performed.
Without loss of generality for the goal of the study, optical
density (OD) values below 0.05 have been considered
as no total cell density/no biofilm formation and have
not been taken into account for the ANOVAs purposes
(Additional file 2: Table S2). From the results of the
ANOVA tests, the culture medium, the incubation
temperature and the interaction were highly significant
for the total cell density and biofilm formation.
Clearly, rich media enhanced not only the growth but
also biofilm production. However, higher total cell dens-
ities were not necessarily correlated with higher amounts
of slime (Table 1). These rich media contain also high
amounts of salts, particularly SASW, for which a marked
shift in biofilm production is observed (Additional file 2:
Table S2, Figure 2B). Indeed, salts increase protein adsorp-
tion on material surfaces, thus facilitating the establish-
ment of the extracellular polymeric substances (EPS) that
constitute the biofilm matrix [42]. Salt ions –cations,
particularly-, are known to play important functions in
the biofilm formation process of halophylic bacteria as
their cytoplasmic enzymes are adapted to high ionic
strengths and consequently they have a requirement of
charged ions to function properly [43]. In this context,
Ca2+ has been observed to be a requirement for the
maintenance of biofilm integrity in certain variants of
Vibrio cholerae and other marine Vibrio species [44], and
a triggering factor of the synthesis of class II proteins
in Pseudoalteromonas sp., which are expressed only in
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Figure 2 Growth and biofilm formation under different conditions. (A) Growth of S. algae CECT 5071 in different media after an incubation
period of 24 h at 26°C and 32°C. (B) Biofilm formation under the same conditions. Bar heights represent the mean of eight replicates. Errors are
expressed as ± standard deviation (SD). Asterisks indicate significant differences for Bonferroni post-hoc comparisons between both incubation
temperatures for the growth medium, and they are shown above the bar with the highest OD in each case. OD values below 0.05 at any
temperature have not been taken into account for statistical purposes.

biofilm cells [45]. In Pseudomonas fluorescens, Mg2+
cation increase cell attachment and condition the
structure and further development of the biofilms [46].
Cations such as Ca2+, Mn2+, Cu2+ or Zn2+ have also been
found to be essential for the formation of air-liquid inter-
face biofilms in Shewanella oneidensis [47]. In fact, when
MH2 is supplemented with 20 mg/L Ca2+ and 10 mg/L
Mn2+ (CAMH2 medium), a shift in biofilm production is
observed (Figure 2B).
The temperature also exerted a significant influence
on both growth and biofilm formation in function of the
medium (Figure 2). The influence of the temperature on
bacterial biofilm formation is usually species and strain-
dependent. For example, Stauder et al. [48] positively
correlated biofilm production and temperature in a V.
cholerae strain, suggesting that higher seawater tempera-
tures increase the persistence of the bacterium in the
aquatic environment. Similarly, Chiu et al. [49] associated
changes in the planktonic and biofilm bacterial communi-
ties with seasonal variations in water temperature and
salinity. In a different study, McDougald et al. [50] found
no correlation between temperature and biofilm formation
in clinical and environmental strains of Vibrio vulnificus,
whereas previous work showed a direct correlation between
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Table 1 Relative biofilm formation for each medium and Table 2 IC50 values for three antifouling biocides towards
temperature S. algae CECT 5071
Culture medium S. algae Culture Inoculum IC50 (μM)
medium size
26°C 32°C TBTO Tralopyril Zinc pyrithione
MB 1.58 ± 0.26 1.14 ± 0.26 MB H 7.8 ± 1.3 14.6 ± 5.8 17.6 ± 1.1
MH2 0.91 ± 0.14 0.33 ± 0.16 S 8.1 ± 1.5 15.8 ± 2.7 13.8 ± 2.0
CAMH2 0.92 ± 0.21 0.75 ± 0.30 D 12.0 ± 2.3 19.9 ± 7.3 35.4 ± 6.1
BHI2 1.64 ± 0.77 1.66 ± 0.77 MH2 H 10.7 ± 0.6 12.8 ± 3.5 12.8 ± 2.6
TSB2 0.32 ± 0.05 0.59 ± 0.17 S 10.3 ± 0.3 16.0 ± 1.8 18.9 ± 1.7
LMB 0.97 ± 0.22 0.56 ± 0.07 D 12.4 ± 1.1 14.9 ± 3.4 16.7 ± 3.3
SASW 8.60 ± 0.84 6.03 ± 0.55 LMB H 8.4 ± 0.5 1.9 ± 0.4 16.7 ± 2.5
VNSS 3.33 ± 0.36 3.99 ± 0.70 S 9.0 ± 0.3 2.5 ± 1.4 22.7 ± 6.5
MMM - - D 10.6 ± 1.4 2.0 ± 0.9 23.2 ± 6.6
Values indicate the mean ± SD (n = 8) OD590/OD625 ratios. OD values below SASW H 9.5 ± 0.4 18.0 ± 1.9 6.0 ± 0.4
0.05 were considered as no growth/no biofilm formation and have not been
taken into account (indicated by a dash). S 11.4 ± 0.3 16.7 ± 0.9 7.8 ± 1.9
D 12.8 ± 0.5 17.3 ± 1.6 7.8 ± 0.7
temperature, salinity and biofilm formation in the same Data (mean ± SD, n = 3) are arranged in function of the culture medium and
bacterial species [51]. In that case, those findings were the initial cell density in each case. S = Standard inoculum; H = Half standard
inoculum; D = Double standard inoculum.
attributed to strain differences.

The IC50 of model antifouling biocides on Shewanella algae
is influenced by the culture medium and the starting
cell density
There is clear evidence that the characteristics of the
growth medium as well as the inoculum size may have a
great influence on the results obtained from susceptibility
tests [52-54]. To explore the effect of these two parame-
ters, changes in the half-maximal inhibitory concentration
(IC50) of three model biocides on S. algae were studied:
the banned TBTO, a metal-based antifouling agent (zinc
pyrithione) and a non-metal antifoulant (tralopyril). Three
initial cell densities were employed: the standard inoculum
size (S) prepared as described in the methods section as
well as half and double this amount (H and D, respect-
ively). Also, four growth media: MB, LMB, SASW and
MH2 were selected. In these media S. algae presented
different growth values and total biofilm production
(Table 1).
Inoculum sizes were determined by plate counts (H =
3.5 ± 0.6 × 105 cfu/ml, n = 4; S = 7.0 ± 0.8 × 105 cfu/ml,
n = 4; D = 1.5 ± 0.6 × 106 cfu/ml, n = 4). The stock solu-
tions of the biocides were prepared in dimethylsulfoxide
(DMSO). The maximum percentage of DMSO inside a
well was 0.25%. At this concentration, no growth inhib-
ition was observed. Table 2 summarises the results ob-
tained in this experiment.
The effect of the culture medium and the inoculum
size on the IC50 were assessed by two-way ANOVAs.
For TBTO, the culture medium and the inoculum size
were highly significant, and no interaction was detected.
For tralopyril only the culture medium was highly sig-
nificant. Finally, for zinc pyrithione the culture medium,
the inoculum size and the interaction were highly
significant.
In spite of the diversity of conditions employed in bac-
terial antifouling bioassays in terms of inocula and media
[5-11], the comparative effect of these conditions on the
activity of model antifouling molecules has been poorly
evaluated. The need for reproducible positive controls to
validate the assays has been underlined previously [55].
Research in other areas with bacteria that require par-
ticular growth conditions such as lactic acid bacteria has
highlighted the influence of the culture conditions on
the activity of antibiotic standards [56,57]. The results
obtained for S. algae show a dependence of the IC50 of
antifouling biocides on small variations in the inoculum
size and on the use of different culture media, which
emphasizes the need for a consensus in this regard. A
tempting alternative would be the adaptation of CLSI
standards for antimicrobial susceptibility testing to the
requirements of biofouling-representative bacteria. It is
interesting to note that biofouling is a phenomenon of
biological adhesion and consequently, growth inhibition
may not be the main endpoint for biological assays [55].
Consequently, conditions a) supporting bacterial growth,
b) promoting biofilm formation and c) mimicking a salt-
rich environment would be desirable.
Shewanella algae biofilms developed in different media
exhibit medium-dependent morphological and
nanomechanical properties
The final step in this study sought the answer for two
questions: i) how is S. algae biofilm structure affected by
the culture medium? and ii) how these different nutrient
Martín-Rodríguez et al. BMC Microbiology 2014, 14:102
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environments affect the mechanical properties of the
biofilms? For this purpose, CLSM and AFM analysis
were conducted on 24-hour S. algae biofilms developed
in the four selected media. In order to respect exactly
the same substrate as that employed for the initial
in vitro experiments, the bottom of the wells of a micro-
titer plate were mechanically sectioned, sterilised, and
used to develop the bacterial biofilms.
CLSM analysis revealed significant differences in biofilm
thickness, surface coverage and morphology (Table 3,
Figure 3, Additional file 3: Figure S1 and Additional file 4:
Table S3). S. algae biofilms reached almost 30 μm thick in
SASW (Figure 3D, Additional file 3: Figure S1D). Simi-
larly, biofilms developed in LMB and MB surpassed the
20 μm thick, even though the surface coverage was not-
ably lower in MB (Figures 3A and C, Additional file 3:
Figures S1A and C). A completely different structural
pattern was observed in MH2. In this medium, S. algae
developed comparatively thin biofilms, reaching a max-
imum of 13.5 μm, but exhibiting a comparatively high sur-
face coverage (Figure 3B, Additional file 3: Figure S1B).
Roughness coefficients are indicative of the degree of
heterogeneity of the biofilms [58]. In fact, these values
(Table 3), which are significantly different in function of the
medium in which the biofilms were formed (Additional
file 4: Table S3) agree with the visual evidence (Figure 3),
and indicate a patchy, heterogeneous biofilm development
in MB and SASW, and more uniform biofilm layers in
MH2 and LMB.
Thus, two trends were observed in biofilm develop-
ment depending on the medium: a clear trend to a
three-dimensional growth, with a variable degree of
homogeneity, in MB, LMB and SASW, and a relatively
horizontal development in MH2, maximising cell-to-cell
and cell-to-substrate interactions. According to this depic-
tion, we will focus on the comparison between MB and
MH2 since they have been considered representative
enough of the two biofilm growth behaviours.
First of all, in order to show the topographic features
exhibited by the studied cells at high resolution, the
samples were imaged in air after being rinsed and dried.
Thus, Figure 1A shows a representative picture of some S.
algae cells attached to the treated polystyrene substrate.
Since these images were obtained in air, some flagella
Table 3 Average values of different biofilm properties in the four selected media
Medium Mean thickness (μm) Max. thickness (μm)
MB 11.2 ± 0.8 25.3 ± 2.3
MH2 9.0 ± 1.2 13.5 ± 1.0
LMB 15.4 ± 2.2 20.5 ± 3.4
SASW 13.0 ± 0.8 29.5 ± 1.9
Biofilm thickness (n = 12), surface coverage (n = 12) and roughness coefficients (n = 12) were determined from CLSM reconstructions. Young modulii and adhesion
forces were quantified by AFM. In this case, at least 115 bacteria were individually analysed for each magnitude. Data represent the average ± SD.
Page 6 of 14
belonging to neighbouring bacteria adsorbed on the
surface could be observed as well. Bacterial cells were 2.2-
3.5 μm in length and 0.4-0.7 μm in width. Some polishing
lines resulting from the disc’s surface treatment are also
visible. Additionally, in Figure 1B, some of these features
can be observed in more detail, namely some flagella
(white arrow), topographic details of the bacterial surface
and submicrometer particles of EPS.
Figures 4A-B correspond to AFM topographic images
recorded in 0.22 μm filtered seawater (FSW) obtained in
MB and MH2, respectively. Nevertheless, supplementary
information (Additional file 5: Figures S2, Additional file 6:
Figure S3, Additional file 7: Figure S4, Additional file 8:
Figure S5, Additional file 9: Figure S6 and Additional
file 10: Figure S7) contains the AFM results obtained for
all the media studied. Three-dimensional aggregates formed
by bacteria linked to each other can be seen in MB, leaving
large bacteria-free areas. Conversely, in Figure 4B, the sub-
strate appears to be covered by a near continuous and
homogenous layer of bacteria and EPS. In this case, three-
dimensional aggregates are present in a remarkably lower
degree. These results revealed a different interaction be-
tween the substrate and the bacterial envelope in function
of the culture medium. Thus, in MH2, bacteria-substrate
interaction is clearly favoured in comparison to MB.
On the other hand, Figures 4C-D compare the Young’s
modulus and Figures 2E-F the adhesion force quantitative
mappings of the same surface area for MB and MH2. In
this context, it should be taken into account that the
greater the brightness of the patches the larger corre-
sponding values of the magnitudes analysed. In general
terms, images show that the higher values in Young’s
modulus and adhesion force correspond to the bacteria-
free substrate areas. Note that the higher pikes present in
the cross sections (E > 0.7 MPa) are related to contributions
due to bacteria/EPS-free substrate. Thus, Young’s modulus
exhibited by bacteria resulted to be significantly larger for
those grown in MH2 (Additional file 4: Table S3). However,
regarding adhesion forces, the situation was exactly the
opposite with the higher figures corresponding to MB.
In addition, by considering the average size of certain
Young’s modulus spots, especially those associated with
clusters of bacteria present in the topographic image, it
can be concluded that these groups of bacteria seem to
Coverage (%) Roughness coefficient Young modulus (MPa) Adhesion (nN)
15.9 ± 1.7 1.92 ± 0.06 0.16 ± 0.10 1.33 ± 0.38
20.9 ± 2.4 0.97 ± 0.15 0.34 ± 0.16 0.73 ± 0.29
32.1 ± 4.6 0.65 ± 0.18 0.22 ± 0.13 0.85 ± 0.35
23.9 ± 3.9 1.40 ± 0.24 0.19 ± 0.09 1.11 ± 0.41
Martín-Rodríguez et al. BMC Microbiology 2014, 14:102
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A B
Figure 3 Effect of the medium on biofilm structure evidenced by CLSM. Projections (upper row) and sections (bottom row) of 24-h S. algae
CECT 5071 biofilms (40x) developed in different media. Columns: (A) MB; (B) MH2; (C) LMB; (D) SASW.
be surrounded by EPS which spreads to the cell-substrate
interface (see also Additional file 6: Figure S3A-F).
Table 3 shows the averaged values of Young’s modulus
and adhesion forces recorded for individual bacterial cells
grown in the four different media. Our overall experimen-
tal data (see histograms in Additional file 8: Figure S5)
confirmed the trend previously described a clear correl-
ation between the rising in Young’s modulus and the
diminishing in the adhesion response is exhibited when
modifying the growth medium. As shown in Table 3,
values registered for MH2 almost doubled those grabbed
for MB. Anyway, the biofilm developed in MH2 showed
the highest elasticity values registered. It should be noted
that these results obtained for the elasticity properties of
the external covering layer of S. algae cells are in the same
order of magnitude as those reported for other gram-
negative bacteria [59,60]. In fact, significant variations in
nanomechanical and physicochemical properties have also
been reported for Escherichia coli and S. putrefaciens cells
depending on the culture conditions and on the pH,
respectively [61,62].
Average adhesion forces are shown in Table 3. As dis-
cussed before, an opposite correlation among data for
Young’s modulii is observed. Thus, figures obtained for
MH2 were significantly lower than those obtained for MB
(Additional file 4: Table S3). Regarding this point, it
should be noted that whereas Young’s modulus is directly
dependent on the mechanical behaviour of the outer part
of the bacteria under tip indentation, adhesion forces
imply specific attractive interactions with the tip. In this
sense, it is worth noting that the abovementioned correl-
ation has not necessarily to be like that.
Although AFM tips have not been functionalised and
consequently the adhesion force response recorded is
due to non-specific interactions [63], it should be noted
Page 7 of 14
C D
that AFM tips, bacteria and incubation times remained
unchanged in all the experiences carried out. Therefore,
differences observed for the biofilms grown in the differ-
ent media reflect unambiguously a significant impact on
the physicochemical properties of biofilms. Consequently,
these results allow us to conclude the substantial effect of
modifying the culture medium on the nanomechanical
and physicochemical behaviour exhibited by the resulting
biofilms.
AFM force-distance curve analysis has also been car-
ried out in order to assess kB, the spring constant of the
bacteria, which eventually resulted also dependent on the
growth medium. Thus, Figure 5A shows representative
force-distance curves registered in seawater for a stiff
surface, mica (black line), and for representative single
deformable bacteria grown in MB (red) and in MH2 (dark
green). In this context, considering the relevant differences
exhibited by the indentation depths grabbed for MB and
MH2, a differential elasticity response can be easily con-
cluded. Indeed, envelope belonging to bacteria grown in
MH2 showed noticeable more rigid profiles than those
corresponding to MB (Figures 5B-C).
By combining properly the Hertz’s model and Hooke’s
law, nanomechanical properties of the bacterial envelope
can be deduced from the experimental loading force-
indentation curves. Thus, according to the Hertzian
model that takes into account the geometry of the tip, the
non-linear regime corresponding to the initial part of the
indentation curve was fitted to evaluate the Young’s
modulus of the bacteria indented [62], green lines in
Figures 5B-C. Likewise, from the linear part of the curve
obtained from experimental data registered at high
loading forces, the kB of the bacteria could be calculated
by linear fitting (magenta lines in Figures 5B-C) [59].
Elasticity values obtained were 0.15 ± 0.08 MPa for MB
Martín-Rodríguez et al. BMC Microbiology 2014, 14:102
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Figure 4 Representative cross-section of 2D peak force tapping 50 x 50 μm2 images. (A) and (B), topographic images of MB and MH2,
respectively, in brown; (C) and (D), Young’s modulus quantitative, in gold; (E) and (F), adhesion forces, grey.
and 0.38 ± 0.11 MPa for MH2. As expected, Young’s
modulus data resulted to be in very good agreement
with those previously obtained by PF-QNM (Table 3).
On the other hand, kB values, which ranged from 0.022
N/m to 0.050 N/m, are consistent with those obtained
for other gram negative bacteria as thoroughly reported
[59,61]. Moreover, these figures exhibited the same trend
showed by elastic modulus when altering the culture
medium.
Conclusions
The influence of the culture medium and the incubation
temperature on the total cell density and biofilm forma-
tion of Shewanella algae CECT 5071 has been studied.
The influence of both factors was found to be highly
significant. Additionally, the culture medium and the in-
oculum size exerted a significant influence on the values
Page 8 of 14
obtained for the IC50 of three antifouling biocides. An
approach to the unification of criteria in antifouling bio-
assays involving marine bacteria could be the adaptation
of already existing, universally-accepted methodologies
to the requirements of test organisms concerning marine
biofouling. With regard to bacteria, CLSI guidelines con-
stitute the most evident and clear reference.
With this work we have established and characterised
in detail a biofilm model for antifouling bioassays. Using
S. algae CECT 5071 as model organism, we were able to
demonstrate quantitatively the influence that the culture
medium exerts not only on the biofilm density or thick-
ness, but more importantly, on the biofilm structure and
on its nanomechanical and physicochemical properties.
CLSM showed two clear architectural patterns in function
of the medium in which the biofilms were developed.
From PF-QNM and FD-AFM data it is possible to infer
Martín-Rodríguez et al. BMC Microbiology 2014, 14:102 Page 9 of 14
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Figure 5 Representative force-distance curves. (A) Representative force-piezo displacement measured on mica (black) and on top of single
bacteria grown in MH2 (dark green) and in MB (red), obtained in seawater. Loading force-indentation depth (blue) curves resulted from subtracting
black curve from the green (B) and the red ones (C) at constant loading force. Curves (B) and (C) were fitted according Hertz’s model (green) and
linear model (magenta) to calculate elasticity modulus and kB, respectively.

that S. algae cells grown in MH2 medium exhibited a Culture media

more complex outer surface, remarkably stiffer and with a
significant higher range of Young’s modulus figures distri-
bution, when compared to the other media which showed
more similar features in this sense. On the other hand,
adhesion forces results evolved in the opposite way thus
confirming the differential physicochemical behaviour
exhibited by the biofilms in function of the nutrient
environment.
Methods
Strains and assay platform
Shewanella algae CECT 5071 was acquired from the
Spanish Type Culture Collection (CECT). The strain
was cryopreserved at −80°C. Before each experiment,
an agar plate was streaked and incubated for 24 h. A
single, isolated colony was selected to streak a second
agar plate that was incubated for other 24 h. Inocula
were prepared from these second agar plates. The
experiments were conducted in 96-well flat-bottom
surface-treated polystyrene microtiter plates (Nunc
167008). For biofilm experiments, the bottom of the
wells were used as substrate (see Microscopy: general
procedures section).
Bacterial growth and biofilm formation were quantified
in nine different media: Marine Broth (MB) (Conda);
Mueller-Hinton Broth (Scharlau) supplemented with
NaCl to give a final concentration of 2% (MH2); cation-
adjusted MH2 (CAMH2), that consisted in MH2 supple-
mented with 55 mg/l CaCl2 and 40 mg/l MgCl2; Brain
Heart Infusion (Scharlau) supplemented with NaCl to a
final concentration of 2% (BHI2); Tryptic Soy Broth (BD)
supplemented with NaCl to a final concentration of 2%
(TSB2); Luria Marine Broth (LMB); Supplemented Arti-
ficial Seawater (SASW); Väätänen Nine-Salt Solution
(VNSS); and Marine Minimal Medium (MMM). LMB
and SASW were prepared according to Lang et al. [35],
NSS and VNSS followed the recipe described by Mårdén
et al. [64]; and MMM was prepared as described by
Östling et al. [65]. A summary of the composition of each
medium is provided as additional information (Additional
file 1: Table S1).
Assessment of growth and biofilm production
Each well of the microtiter plate contained 100 μl of
bacterial inoculum and 100 μl the appropriate culture
medium. Growth at two temperatures (26 and 32°C) was
Martín-Rodríguez et al. BMC Microbiology 2014, 14:102
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quantified after an incubation period of 24 h by measuring
the optical density at 625 nm (OD 625) with an automatic
plate reader (Perkin-Elmer EnSpire). Eight replicates were
used for each medium.
Once the growth was measured, biomass was quantified
by the crystal violet (CV) staining method [66]. Briefly,
wells were thoroughly washed three times with water to
remove the culture medium and planktonic cells as well
as loosely adhered bacteria. Firmly attached bacteria were
heat fixed (65°C) for 30–45 min and then 200 μL of a
0.2% CV solution (Sigma-Aldrich) were added to each
well. After 15 min wells were emptied and washed care-
fully with water. Plates were air-dried and then the dye
was solubilised by addition of 200 μl of absolute ethanol.
Absorbance was recorded at 590 nm. When OD590 read-
ings were above 2.5, the sample was tenfold diluted and
OD was measured again [67].
Three classic antifouling agents: TBTO, tralopyril and
zinc pyrithione were purchased from Sigma-Aldrich.
Stock solutions of the products (40 mM) dissolved in
dimethylsulfoxide (DMSO) were diluted in the culture
medium to give a final test concentration of 100 μM.
Serial dilutions (100, 50, 10, 5, 1, 0.5, 0.1 and 0.05 μM)
were performed for the determination of the IC50 in MB,
MH2, LMB and SASW. OD readings were normalised
with respect to the absorbance of the blank wells and then
the growth inhibition percentage respect to a control with
the proportional amount of DMSO was calculated. Exper-
iments were run by triplicate.
Preparation of inocula
Bacterial inocula were prepared in 0.22 μm filtered sea-
water (FSW). Isolated colonies were suspended until
they matched a McFarland turbidity of 0.5 (bioMérieux
Vitek Densichek). One hundred microliters were trans-
ferred to test tubes containing 9.9 ml of the appropriate
culture medium. This cell suspension constituted the
standard starting inoculum (S) as defined by CLSI guide-
lines for antimicrobial susceptibility testing [68]. Double
(D) and half (H) the size of the standard inoculum were
used to evaluate the effect of the initial cell density on the
activity of biocides towards S. algae. To check the actual
starting cell number, a 200 μl sample of the inoculum was
serially tenfold diluted from 10−1 to 10−8. Four 10 μl drops
from each dilution were spotted on agar plates and incu-
bated. Colony formation was assessed after 24 h.
Microscopy: general procedures
For microscopy experiments, the bottoms of the wells
of a microtiter plate were mechanically sectioned with
a computer numerical control milling machine (Fagor
CNC 8055 M) in order to use exactly the same substrate
as in previous tests. The sectioned discs thus obtained
(5.86-5.98 mm in diameter, 1.00-1.08 mm in height, data
Page 10 of 14
from 15 random measurements) were carefully disengaged
and sterilised by a brief sonication in ethanol and UV
irradiation before their use in the experiments. To develop
the biofilms, the discs were placed at the bottom of a
24-well microtiter plate. Two-mililiter bacterial cultures
were prepared in the appropriate medium following the
same procedures as described previously. After the incu-
bation period, discs were rinsed three times with FSW and
kept immersed upon their use in the microscope.
Confocal Laser Scanning Microscopy
Biofilms formed on polystyrene discs were fluorescently
stained with acridine orange (AO), a membrane permeant
nucleic acid stain that intercalates dsDNA and binds to
ssDNA as well as to ssRNA through dye-base stacking to
give broad spectrum fluorescence when excited at 476 nm
[69]. This compound stains all cells in a biofilm, live or
dead, and may also bind to nucleic acids that are present
in the extracellular matrix. To stain biofilms, discs were
immersed in 0.1% w/v AO (Sigma-Aldrich) in PBS for 5
min at room temperature and washed with FSW. Fluores-
cently labelled biofilms were placed in two drops of 0.9%
FSW on the surface of a glass coverslip and were exam-
ined using an Olympus Fluoview 1000 Confocal Laser
Scanning Microscope. Each biofilm was scanned at 4 posi-
tions randomly selected at the microscope stage and
confocal image series were generated by optical sectioning
at each of these positions. Three independent biofilm ex-
periments were performed, and image stacks of 512×512
pixels were collected for quantification. Image combining
and processing were performed with the Imaris software
package, version 4.0 (Bitplane AG, Zürich, Switzerland).
The biofilm structure was quantified using the software
program COMSTAT [70] available as free download-
able software at http://www.imageanalysis.dk. COMSTAT
converts pixels from confocal image stacks into numerical
values, facilitating quantitative characterization of each
structural component within 3D biofilm images [71].
COMSTAT software allowed the determination of:
– Mean thickness: the average thickness, from the base
at the biofilm–substratum interface to the top of the
biofilm in the channel lumen, across the entire biofilm
in the field of view. Mean biofilm thickness provides a
measure of the spatial size of the biofilm.
– Maximum thickness: the maximum thickness over a
given location, ignoring pores and voids inside the
biofilm.
– Roughness coefficient: a measure of variation in
biofilm thickness across the field of view, an
indicator of biofilm heterogeneity.
The percentage of adhering cells (% Coverage) was cal-
culated using ImageJ NIH image processing software [72].
Martín-Rodríguez et al. BMC Microbiology 2014, 14:102
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Atomic Force Microscopy
Imaging and force measurements to characterise the
nanomechanical properties of Shewanella algae cells were
performed by AFM. In these studies every treated polystyr-
ene disc containing the immobilised bacteria was attached
to a steel sample puck by means of an adhesive tape. When
measuring in liquid, 50 μL of FSW were added onto the
disc prior to be placed into the AFM liquid cell. For mea-
surements performed in air, polystyrene discs were carefully
rinsed and dried in N2 atmosphere before using.
Tapping Mode: S. algae cells were imaged by AFM op-
erating in tapping mode in air using a Multimode micro-
scope and a Nanoscope V control unit from Bruker at a
scan rate of 1.0–1.2 Hz. To this end, etched silicon tips
(RTESP, 271–311 kHz, and 40–80 N/m) were used.
Peak Force Tapping and force-distance analysis: Quantita-
tive mapping were performed in FSW at room temperature
using a Nanoscope V controller (Bruker). Images were
acquired in AFM contact and Peak Force Tapping Mode
[73] (Peak Force-Quantitative Nanomechanics, PF-QNM).
AFM probes used in these studies were silicon nitride
probes (NP-C, Bruker) with a nominal tip radius of 20–60
nm. The spring constant of cantilevers were measured
using the thermal tuning method [74], and its values
ranged 0.14-0.26 N/m. Mica surfaces were selected as
rigid substrates for deflection sensitivity calibration. Note
that in PF-QNM measurements AFM tips were carefully
calibrated before every experience as described elsewhere
[74-77]. Experimental results were acquired for single bac-
teria or little groups of them from the PF-QNM images,
excluding thus contributions due to bacteria/EPS-free
substrate. Data proceeding from at least 115 units from
two independent cultures were collected for each medium.
Adhesion force and Young’s modulus values distribution
has been expressed as histograms. Force-distance (FD)
curves were collected using low loading forces (F < 20 nN)
in order to protect both the AFM tip and the bacterial cells
[59]. Data processing was carried out using the commercial
Nanoscope Analysis (Bruker), WSxM (Nanotec) [78] and
Gwyddeon (GNU) softwares.
Statistics
The effects of culture medium, incubation temperature
and their interaction on the dependent variables (total
cell density and biofilm formation) were assessed by a
two-way ANOVA. The effects of culture medium, the
inoculum size and their interaction on the IC50 were
assessed by two-way ANOVAs for different biocides with
S. algae. In all cases a balanced design was performed
and a fixed-effects model of analysis of variance was
applied. In some cases the response variable was square
root transformed to improve homocedasticidy. Bartlett test
was performed to check this assumption and normality
was verified by means of Kolmogorov-Smirnoff test for
Page 11 of 14
residuals. Tukey or Bonferroni multiple comparison post
hoc tests were assessed in all the instances. The IBM®
SPSS® Statistics 19.0 was used for statistical analysis. A
significance level at 0.05 was set.
To assess the effect of culture medium on S. algae biofilm
structure, a one way ANOVA for each of the following
variables: mean and maximum thickness, coverage and
roughness coefficient, were performed, followed by a
Tukey test to check for differences between the four
culture media. Mean thickness was logarithmic trans-
formed to improve homocedasticity. Moreover, the effect
of culture medium on the Young’s modulus and adhesion
force, both of them normally distributed but with unequal
variances, was conducted by means of a Welch one-way
ANOVA followed by a Games Howell post hoc test.
For all the variables, the culture medium was highly
significant.
Half-maximal inhibitory concentration values (IC50) were
determined with GraphPad Prism 5 using a four-parameter
non-linear regression model (GraphPad Software Inc., La
Jolla, CA, USA).
Additional files
Additional file 1: Table S1. Media composition. A detailed list of the
components of each medium is provided (g/l).
Additional file 2: Table S2. Two-way ANOVA test design and results
for the growth and biofilm formation experiments. Two-way ANOVA was
conducted with total cell density and biofilm formation as dependent
variables and two factors, culture medium and incubation temperature.
The dependent variable has been square-root (SR) transformed to ensure
homocedasticity.
Additional file 3: Figure S1. Detail of biofilm thickness in each
medium. (A) MB; (B) MH2; (C) LMB; (D) SASW.
Additional file 4: Table S3. One-way ANOVA and Welch ANOVA results
for CLSM and AFM data, respectively. For the one-way ANOVA, the dependent
variable has been logarithmic transformed to ensure homocedasticity.
Additional file 5: Figure S2. Representative 3D Peak Force Tapping 50
x 50 μm2 images of Shewanella algae grown in different nutritive media.
(A) MB; (B) MH2; (C) LMB; and (D) SASW.
Additional file 6: Figure S3. Representative 3D Peak Force Tapping 50
x 50 μm2 images (A)-(D), topographic images corresponding to media
MB, MH2, LMB, and SASW, respectively, in brown; (E)-(H), Young’s
modulus quantitative mappings, in gold; (I)-(L), adhesion forces, grey.
Additional file 7: Figure S4. Representative cross-sections of 2D Peak
Force Tapping 50 x 50 μm2 images. (A) and (B), topographic images of
media LMB and SASW, respectively, in brown; (C) and (D), Young’s modulus
quantitative mappings, in gold; (E) and (F), adhesion forces, grey.
Additional file 8: Figure S5. Histograms showing the elastic modulus
(E, red bars) and adhesion force (blue) distributions for Shewanella algae
cells. (A) and (E) MB; (B) and (F) MH2; (C) and (G) LMB; (D) and (H) SASW.
Additional file 9: Figure S6. Representative cross-section of 2D Peak
Force Tapping 15 x 15 μm2 images. (A)-(B), topographic images of media
MB, MH2, LMB, and SASW, respectively, in brown; (E)-(H), Young’s modulus
quantitative mappings, in gold; (I)-(L), adhesion forces, grey.
Additional file 10: Figure S7. Representative 2D Peak Force Tapping
2.7 x 2.7 μm2 (upper panel) and 4.5 x 4.5 μm2 (lower panel) images. (A)
and (B), topographic images of media MB and MH2, respectively, in
brown; (C) and (B), Young’s modulus quantitative, in gold; (E) and (F),
adhesion forces, grey.
Martín-Rodríguez et al. BMC Microbiology 2014, 14:102
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
AJM-R and JJF conceived and designed the experiments. AJM-R conducted
the experiments. AG-O and AH-C conducted the AFM work and processed
the results from AFM measurements. AM conducted the CLSM work. RD-G
carried out the statistical analysis. VSM and MN contributed with reagents,
materials and valuable advice in the experimental design. AJM-R, AG-O, AH-C
and JJF analysed the data. AJM-R and JJF wrote the paper. All authors read
and approved the final manuscript.
Acknowledgements
Financial support was provided by grants from the Spanish Ministry of
Economy and Competitiveness (MINECO): SAF2011-28883-C03-01,
CTQ2011-28417-C02-01/BQU, CTQ2011-24784, MTM2010-16828, and
FP7-EU: REGPOT-2012-CT2012-316137-IMBRAIN. AJM-R acknowledges
PLOCAN for the grant received. A.G-O. thanks Fundación CajaCanarias for
a SEGAI grant. Dr. Basilio Valladares is acknowledged for the use of the
facilities at the University Institute of Tropical Diseases and Public Health
of the Canary Islands.
Author details
1
Institute for Bio-Organic Chemistry “Antonio González”, Center for Biomedical
Research of the Canary Islands (CIBICAN), University of La Laguna, Avenida
Astrofísico Francisco Sánchez 2, La Laguna, Tenerife 38206, Spain. 2Department
of Physical Chemistry, University of La Laguna, Avenida Astrofísico Francisco
Sánchez 1, La Laguna, Tenerife 38206, Spain. 3Department of Physiology,
Institute of Biomedical Technologies, CIBICAN, University of La Laguna, Campus
de Ofra, s/n, La Laguna, Tenerife 38071, Spain. 4Department of Statistics,
Operations Research and Computation, University of La Laguna, Avenida
Astrofísico Francisco Sánchez 2, La Laguna, Tenerife 38206, Spain. 5Oceanic
Platform of the Canary Islands, Carretera de Taliarte s/n, Telde, Gran Canaria
35214, Spain. 6Institute of Materials and Nanotechnology, University of La
Laguna, Avenida Astrofísico Francisco Sánchez s/n, La Laguna, Tenerife 38206,
Spain.
Received: 22 December 2013 Accepted: 11 April 2014
Published: 23 April 2014
References
1. Ortlepp S, Pedpradap S, Dobretsov S, Proksch P: Antifouling activity of
sponge-derived polybrominated diphenyl ethers and synthetic
analogues. Biofouling 2008, 24:201–208.
2. Lejars M, Margaillan A, Bressy C: Fouling release coatings: a nontoxic
alternative to biocidal antifouling coatings. Chem Rev 2012,
112:4347–4390.
3. Almeida E, Diamantino TC, de Sousa O: Marine paints: the particular case
of antifouling paints. Prog Org Coatings 2007, 59:2–20.
4. Chambers LD, Stokes KR, Walsh FC, Wood RJK: Modern approaches to
marine antifouling coatings. Surf Coatings Technol 2006, 201:3642–3652.
5. Maréchal J-P, Culioli G, Hellio C, Thomas-Guyon H, Callow ME, Clare AS,
Ortalo-Magné A: Seasonal variation in antifouling activity of crude
extracts of the brown alga Bifurcaria bifurcata (Cystoseiraceae) against
cyprids of Balanus amphitrite and the marine bacteria Cobetia marina
and Pseudoalteromonas haloplanktis. J Exp Mar Bio Ecol 2004, 313:47–62.
6. Tsoukatou M, Maréchal JP, Hellio C, Novaković I, Tufegdzic S, Sladić D, Gasić
MJ, Clare AS, Vagias C, Roussis V: Evaluation of the activity of the sponge
metabolites avarol and avarone and their synthetic derivatives against
fouling micro- and macroorganisms. Molecules 2007, 12:1022–1034.
7. Mokrini R, Ben Mesaoud M, Daoudi M, Hellio C, Maréchal J-P, El HM,
Ortalo-magne A, Piovetti L, Culioli G: Meroditerpenoids and derivatives
from the brown alga Cystoseira baccata and their antifouling properties.
J Nat Prod 2008, 71:1806–1811.
8. Plouguerné E, Hellio C, Deslandes E, Véron B, Stiger-Pouvreau V:
Anti-microfouling activities in extracts of two invasive algae: Grateloupia
turuturu and Sargassum muticum. Bot Mar 2008, 51:202–208.
9. Bazes A, Silkina A, Defer D, Bernède-Bauduin C, Quéméner E, Braud J-P,
Bourgougnon N: Active substances from Ceramium botryocarpum used as
antifouling products in aquaculture. Aquaculture 2006, 258:664–674.
Page 12 of 14
10. Bazes A, Silkina A, Douzenel P, Faÿ F, Kervarec N, Morin D, Berge J-P,
Bourgougnon N: Investigation of the antifouling constituents from
the brown alga Sargassum muticum (Yendo) Fensholt. J Appl Phycol
2008, 21:395–403.
11. Qi S-H, Zhang S, Qian P-Y, Wang B-G: Antifeedant, antibacterial, and antilarval
compounds from the South China Sea seagrass Enhalus acoroides. Bot Mar
2008, 51:441–447.
12. Holt HM, Gahrn-Hansen B, Bruun B: Shewanella algae and Shewanella
putrefaciens: clinical and microbiological characteristics. Clin Microbiol
Infect 2005, 11:347–352.
13. Rodrigues JLM, Serres MH, Tiedje JM: Large-scale comparative phenotypic
and genomic analyses reveal ecological preferences of Shewanella
species and identify metabolic pathways conserved at the genus level.
Appl Environ Microbiol 2011, 77:5352–5360.
14. Hau HH, Gralnick J: Ecology and biotechnology of the genus Shewanella.
Annu Rev Microbiol 2007, 61:237–258.
15. El-Naggar MY, Wanger G, Leung KM, Yuzvinsky TD, Southam G, Yang J, Lau
WM, Nealson KH, Gorby Y: Electrical transport along bacterial nanowires
from Shewanella oneidensis MR-1. Proc Natl Acad Sci U S A 2010,
107:18127–18131.
16. Patel P, Callow ME, Joint I, Callow J: Specificity in the settlement –
modifying response of bacterial biofilms towards zoospores of the
marine alga Enteromorpha. Environ Microbiol 2003, 5:338–349.
17. Tait K, Williamson H, Atkinson S, Williams P, Cámara M, Joint I: Turnover of
quorum sensing signal molecules modulates cross-kingdom signalling.
Environ Microbiol 2009, 11:1792–1802.
18. Twigg MS, Tait K, Williams P, Atkinson S, Cámara M: Interference with the
germination and growth of Ulva zoospores by quorum-sensing molecules
from Ulva-associated epiphytic bacteria. Environ Microbiol 2014,
16:445–453.
19. Wahl M, Goecke F, Labes A, Dobretsov S, Weinberger F: The second skin:
ecological role of epibiotic biofilms on marine organisms. Front Microbiol
2012, 3:292.
20. Yang J-L, Shen P-J, Liang X, Li Y-F, Bao W-Y, Li J-L: Larval settlement and
metamorphosis of the mussel Mytilus coruscus in response to
monospecific bacterial biofilms. Biofouling 2013, 29:247–259.
21. Huggett M, Crocetti G, Kjelleberg S, Steinberg P: Recruitment of the sea
urchin Heliocidaris erythrogramma and the distribution and abundance
of inducing bacteria in the field. Aquat Microb Ecol 2008, 53:161–171.
22. Sukovich DJ, Seffernick JL, Richman JE, Hunt K, Gralnick J, Wackett LP:
Structure, function, and insights into the biosynthesis of a head-to-head
hydrocarbon in Shewanella oneidensis strain MR-1. Appl Environ Microbiol
2010, 76:3842–3849.
23. Jiang H-F, Liu X-L, Chang Y-Q, Liu M-T, Wang G-X: Effects of dietary
supplementation of probiotic Shewanella colwelliana WA64,
Shewanella olleyana WA65 on the innate immunity and disease
resistance of abalone, Haliotis discus hannai Ino. Fish Shellfish
Immunol 2013, 35:86–91.
24. Lobo C, Moreno-Ventas X, Tapia-Paniagua S, Rodríguez C, Moriñigo M, de La
Banda IG: Dietary probiotic supplementation (Shewanella putrefaciens
Pdp11) modulates gut microbiota and promotes growth and condition
in Senegalese sole larviculture. Fish Physiol Biochem 2014, 40:295–309.
25. Gram L, Bundvad A, Melchiorsen J, Johansen C: Occurrence of Shewanella
algae in Danish coastal water and effects of water temperature and
culture conditions on its survival. Appl Environ Microbiol 1999,
65:3896–3900.
26. Richards GP, Watson M, Crane EJ, Burt IG, Bushek D: Shewanella and
Photobacterium spp. in oysters and seawater from the Delaware Bay.
Appl Environ Microbiol 2008, 74:3323–3327.
27. Pagani L, Lang A, Vedovelli C, Rimenti G, Pristerà R, Mian P, Moling O,
Pristera R: Soft tissue infection and bacteremia caused by Shewanella
putrefaciens. J Clin Microbiol 2003, 41:2240–2242.
28. Vignier N, Barreau M, Olive C, Baubion E, Théodose R, Hochedez P, Cabié A:
Human infection with Shewanella putrefaciens and S. algae: Report of 16
cases in Martinique and review of the literature. Am J Trop Med Hyg 2013,
89:151–156.
29. Brink AJ, van Straten A, van Rensburg AJ: Shewanella (Pseudomonas)
putrefaciens bacteremia. Clin Infect Dis 1995, 20:1327–1332.
30. Poovorawan K, Chatsuwan T, Lakananurak N, Chansaenroj J, Komolmit P,
Poovorawan Y: Shewanella haliotis associated with severe soft tissue
infection, Thailand, 2012. Emerg Infect Dis 2013, 19:1019–1021.
Martín-Rodríguez et al. BMC Microbiology 2014, 14:102
http://www.biomedcentral.com/1471-2180/14/102
31. Zong Z: Nosocomial peripancreatic infection associated with Shewanella
xiamenensis. J Med Microbiol 2011, 60:1387–1390.
32. Harrison JJ, Stremick C, Turner RJ, Allan ND, Olson ME, Ceri H: Microtiter
susceptibility testing of microbes growing on peg lids: a miniaturized
biofilm model for high-throughput screening. Nat Protoc 2010,
5:1236–1254.
33. Heu C, Berquand A, Elie-Caille C, Nicod L: Glyphosate-induced stiffening of
HaCaT keratinocytes, a Peak Force Tapping study on living cells. J Struct
Biol 2012, 178:1–7.
34. Berquand A, Holloschi A, Trendelenburg M, Kioschis P: Analysis of
cytoskeleton-destabilizing agents by optimized optical navigation and
AFM force measurements. Micros Today 2010, 18:34–37.
35. Lang S, Hüners M, Verena L: Bioprocess engineering data on the
cultivation of marine prokaryotes and fungi. Adv Biochem Eng Biotechnol
2005, 97:29–62.
36. Väätänen P: Effects of composition of substrate and inoculation
technique on plate counts of bacteria in the Northern Baltic Sea. J Appl
Microbiol 1977, 42:437–443.
37. Yee LH, Holmström C, Fuary ET, Lewin NC, Kjelleberg S, Steinberg PD:
Inhibition of fouling by marine bacteria immobilised in kappa-carrageenan
beads. Biofouling 2007, 23:287–294.
38. Castro D, Pujalte MJ, Lopez-Cortes L, Garay E, Borrego JJ: Vibrios isolated
from the cultured manila clam (Ruditapes philippinarum): numerical
taxonomy and antibacterial activities. J Appl Microbiol 2002, 93:438–447.
39. Jorgensen JH, Hindler JF: New consensus guidelines from the Clinical and
Laboratory Standards Institute for antimicrobial susceptibility testing of
infrequently isolated or fastidious bacteria. Clin Infect Dis 2007,
44:280–286.
40. Galkiewicz JP, Pratte Z, Gray M, Kellogg C: Characterization of culturable
bacteria isolated from the cold-water coral Lophelia pertusa. FEMS
Microbiol Ecol 2011, 77:333–346.
41. Moskot M, Kotlarska E, Jakóbkiewicz-banecka J, Fari K, Węgrzyn G, Wróbel B:
Metal and antibiotic resistance of bacteria isolated from the Baltic Sea.
Int Microbiol 2012, 15:131–139.
42. Poleunis C, Rubio C, Compère C, Bertrand P: Role of salts on the BSA
adsorption on stainless steel in aqueous solutions. II. ToF-SIMS spectral
and chemical mapping study. Surf Interface Anal 2002, 34:55–58.
43. Kapfhammer D, Karatan E, Pflughoeft KJ, Watnick PI: Role for glycine
betaine transport in Vibrio cholerae osmoadaptation and biofilm
formation within microbial communities. Appl Environ Microbiol 2005,
71:3840–3847.
44. Kierek K, Watnick PI: The Vibrio cholerae O139 O-antigen polysaccharide is
essential for Ca2+-dependent biofilm development in sea water. Proc Natl
Acad Sci U S A 2003, 100:14357–14362.
45. Patrauchan M, Sarkisova S, Sauer K, Franklin MJ: Calcium influences cellular
and extracellular product formation during biofilm-associated growth of
a marine Pseudoalteromonas sp. Microbiology 2005, 151:2885–2897.
46. Song B, Leff LG: Influence of magnesium ions on biofilm formation by
Pseudomonas fluorescens. Microbiol Res 2006, 161:355–361.
47. Liang Y, Gao H, Chen J, Dong Y, Wu L, He Z, Liu X, Qiu G, Zhou J: Pellicle
formation in Shewanella oneidensis. BMC Microbiol 2010, 10:291.
48. Stauder M, Vezzulli L, Pezzati E, Repetto B, Pruzzo C: Temperature affects
Vibrio cholerae O1 El Tor persistence in the aquatic environment via an
enhanced expression of GbpA and MSHA adhesins. Environ Microbiol Rep
2010, 2:140–144.
49. Chiu JMY, Thiyagarajan V, Tsoi MMY, Qian PY: Qualitative and quantitative
changes in marine biofilms as a function of temperature and salinity in
summer and winter. Biofilms 2006, 2:183–195.
50. McDougald D, Lin WH, Rice S, Kjelleberg S: The role of quorum sensing
and the effect of environmental conditions on biofilm formation by
strains of Vibrio vulnificus. Biofouling 2006, 22:161–172.
51. Joseph LA, Wright AC: Expression of Vibrio vulnificus capsular
polysaccharide inhibits biofilm formation. J Bacteriol 2004,
186:889–893.
52. Egervärn M, Lindmark H, Roos S, Huys G, Lindgren S: Effects of
inoculum size and incubation time on broth microdilution
susceptibility testing of lactic acid bacteria. Antimicrob Agents
Chemother 2007, 51:394–396.
53. Bidlas E, Du T, Lambert RJW: An explanation for the effect of inoculum
size on MIC and the growth/no growth interface. Int J Food Microbiol
2008, 126:140–152.
Page 13 of 14
54. Heindl H, Thiel V, Wiese J, Imhoff JF: Bacterial isolates from the bryozoan
Membranipora membranacea : influence of culture media on isolation
and antimicrobial activity. Int Microbiol 2012, 15:17–32.
55. Briand J-F: Marine antifouling laboratory bioassays: an overview of their
diversity. Biofouling 2009, 25:297–311.
56. Klare I, Konstabel C, Müller-bertling S, Huys G, Vancanneyt M, Swings J,
Goossens H, Witte W, Mu S, Reissbrodt R: Evaluation of new broth media
for microdilution antibiotic susceptibility testing of Lactobacilli,
Pediococci, Lactococci, and Bifidobacteria. Appl Environ Microbiol 2005,
71:8982–8986.
57. Huys G, D’Haene K, Swings J: Influence of the culture medium on
antibiotic susceptibility testing of food-associated lactic acid bacteria
with the agar overlay disc diffusion method. Lett Appl Microbiol 2002,
34:402–406.
58. Murga R, Stewart PS, Daly D: Quantitative analysis of biofilm thickness
variability. Biotechnol Bioeng 1995, 45:503–510.
59. Arnal L, Serra DO, Cattelan N, Castez MF, Vázquez L, Salvarezza RC, Yantorno
OM, Vela ME: Adhesin contribution to nanomechanical properties of the
virulent Bordetella pertussis envelope. Langmuir 2012, 28:7461–7469.
60. Polyakov P, Soussen C, Duan J, Duval JFL, Brie D, Francius G: Automated
force volume image processing for biological samples. PLoS One 2011,
6:e18887.
61. Oh YJ, Jo W, Yang Y, Park S: Influence of culture conditions on Escherichia
coli O157:H7 biofilm formation by atomic force microscopy.
Ultramicroscopy 2007, 107:869–874.
62. Gaboriaud F, Bailet S, Dague E, Jorand F: Surface structure and
nanomechanical properties of Shewanella putrefaciens bacteria at two
pH values (4 and 10) determined by atomic force microscopy. J Bacteriol
2005, 187:3864–3868.
63. Alsteens D, Dague E, Rouxhet PG, Baulard AR, Dufrêne YF: Direct
measurement of hydrophobic forces on cell surfaces using AFM.
Langmuir 2007, 23:11977–11979.
64. Mårdén P, Tunlid A, Malmcrona-Friberg K, Odham G, Kjelleberg S: Physiological
and morphological changes during short term starvation of marine
bacterial islates. Arch Microbiol 1985, 142:326–332.
65. Östling J: Behaviour of IncP-1 plasmids and a miniMu transposon in a
marine Vibrio sp.: isolation of starvation inducible lac operon fusions.
FEMS Microbiol Ecol 1991, 86:83–93.
66. O’Toole GA, Kolter R: Initiation of biofilm formation in Pseudomonas
fluorescens WCS365 proceeds via multiple, convergent signalling
pathways: a genetic analysis. Mol Microbiol 1998, 28:449–461.
67. Kwasny SM, Opperman TJ: Static biofilm cultures of Gram-positive pathogens
grown in a microtiter format used for anti-biofilm drug discovery. Curr Protoc
Pharmacol 2010, Chapter 13:Unit 13A.8.
68. CLSI: Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That
Grow Aerobically; Approved Standard — Ninth Edition. Volume 32. Wayne, PA,
USA: Clinical and Laboratory Standards Institute; 2012.
69. Bernas T, Asem EK, Robinson JP, Cook PR, Dobrucki JW: Confocal
fluorescence imaging of photosensitized DNA denaturation in cell
nuclei. Photochem Photobiol 2005, 81:960–969.
70. Heydorn A, Nielsen AT, Hentzer M, Sternberg C, Givskov M, Ersbøll BK, Molin
S: Quantification of biofilm structures by the novel computer program
COMSTAT. Microbiology 2000, 146:2395–2407.
71. Klein MI, Xiao J, Heydorn A, Koo H: An analytical tool-box for
comprehensive biochemical, structural and transcriptome
evaluation of oral biofilms mediated by mutans streptococci.
J Vis Exp 2011, 47:2512.
72. Schneider CA, Rasband WS, Eliceiri KW: NIH Image to ImageJ: 25 years of
image analysis. Nat Methods 2012, 9:671–675.
73. Dufrêne YF, Martínez-Martín D, Medalsy I, Alsteens D, Müller DJ: Multiparametric
imaging of biological systems by force-distance curve-based AFM.
Nat Methods 2013, 10:847–854.
74. Dokukin ME, Sokolov I: Quantitative mapping of the elastic modulus of
soft materials with HarmoniX and PeakForce QNM AFM modes. Langmuir
2012, 28:16060–16071.
75. Berquand A, Roduit C, Kasas S, Holloschi A, Ponce L, Hafner M: Atomic force
microscopy imaging of living cells. Micros Today 2010, 18:8–14.
76. Pletikapić G, Berquand A, Radić TM, Svetličić V: Quantitative
nanomechanical mapping of marine diatom in seawater using
Peak Force Tapping Atomic Force Microscopy. J Phycol 2012,
48:174–185.
Martín-Rodríguez et al. BMC Microbiology 2014, 14:102 Page 14 of 14
http://www.biomedcentral.com/1471-2180/14/102

77. Alsteens D, Dupres V, Yunus S, Latgé J-P, Heinisch JJ, Dufrêne YF:
High-resolution imaging of chemical and biological sites on living
cells using peak force tapping atomic force microscopy. Langmuir
2012, 28:16738–16744.
78. Horcas I, Fernández R, Gómez-Rodríguez JM, Colchero J, Gómez-Herrero J,
Baro AM: WSxM: a software for scanning probe microscopy and a tool
for nanotechnology. Rev Sci Instrum 2007, 78:013705.

doi:10.1186/1471-2180-14-102
Cite this article as: Martín-Rodríguez et al.: On the influence of the
culture conditions in bacterial antifouling bioassays and biofilm
properties: Shewanella algae, a case study. BMC Microbiology 2014 14:102.

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